Identification, expression profiles, and binding properties of chemosensory protein 18 in Plutella xylostella (Lepidoptera: Plutellidae)

Abstract Chemosensory proteins (CSPs) are highly efficient carry tools to bind and deliver hydrophobic compounds, which play an important role in the chemosensory process in insects. The diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), is a cosmopolitan pest that attacks cruciferous crops. However, the detailed physiological functions of CSPs in P. xylostella remain limited to date. Here, we identified a typical CSP, named PxylCSP18, in P. xylostella and investigated its expression patterns and binding properties of volatiles. PxylCSP18 was highly expressed in antennae and head (without antennae), and the expression level in the male antennae of P. xylostella was obviously higher than that in the female antennae. Moreover, PxylCSP18 has a relatively broad binding spectrum. Fluorescence competitive binding assays showed that PxylCSP18 had strong binding abilities with 14 plant volatiles (Ki < 10 μM) that were repellent or attractive to P. xylostella. Notably, PxylCSP18 had no significant binding affinity to (Z)-11-hexadecenal, (Z)-11-hexadecenyl acetate, and (Z)-11-hexadecenyl alcolol, which are the pheromone components of P. xylostella. The attractive effects of trans-2-hexen-1-ol and isopropyl isothiocyanate to male adults and the attractive effects of isopropyl isothiocyanate and the repellent effects of linalool to female adults were significantly decreased after knocked down the expression of PxylCSP18. Our results revealed that PxylCSP18 might play an important role in host plant detection, avoidance of unsuitable hosts, and selection of oviposition sites; however, it does not participate in mating behavior. Overall, these results extended our knowledge on the CSP-related functions, which provided insightful information about CSP-targeted insecticides.


Introduction
The olfactory system regulates many insect behaviors, including food sources detection, unsuitable host avoidance, selection of oviposition sites, mate choice, and natural enemy avoidance, and plays a pivotal role in the life cycle of insects (Pickett and Woodcock 1996, Field et al. 2000, Riffell and Alarcon 2013, Cao et al. 2020, Wang et al. 2022).As one of the most important olfactory organs, the antennae was covered with many olfactory sensilla that have olfactory sensory neurons (OSNs) (Su et al. 2009, Yan et al. 2017).Volatiles from the environment were received by the olfactory organs, then solubilized and carried by odorant binding proteins (OBPs) or chemosensory proteins (CSPs) to ORNs, and finally, the odor information is transformed into neuronal electrical signals (Pelosi et al. 2006, 2018, Leal 2013).In this process, a wealth of olfaction-related genes were reported to participate in the molecular network of olfaction, such as CSPs, OBPs, odorant-degrading enzymes (ODEs), ionotropic receptors (IRs), olfactory receptors (ORs), sensory neuron membrane proteins (SNMPs), and odorant receptor coreceptor (Orco) (Vogt 2003, Pelosi et al. 2006, 2018, Vogt et al. 2009, Abuin et al. 2011, Leal 2013, Ferguson et al. 2021, Konopka et al. 2021).
Insect CSPs, which have low molecular weight with about 100-120 amino acid residues, are characterized by small size and compact structure that evolutionarily conserved (Pelosi et al. 2006, Fu et al. 2020).The first CSP of insects, OS-D protein, was identified in the antennae of Drosophila melanogaster through subtractive hybridization (McKenna et al. 1994).CSPs were initially reported to be involved in limb regeneration, and later were their function as olfactory proteins (Yi et al. 2014).To date, the information available for CSPs, also known as sensory appendage proteins (SAPs), indicated that these small soluble proteins involved in diverse activities and endowed with heterogeneous functions, such as serving as carrier proteins for binding and delivering hydrophobic compounds in chemosensory process (Robertson et al. 1999, Pelosi et al. 2006, 2018, Fu et al. 2020).Studies on the molecular structures suggested CSPs are constituted by 4 conserved cysteines, which arrangement mode shown as Cys 1 -X6-Cys 2 -X18-Cys 3 -X2-Cys 4 , and linked by disulfide bridges between 2 adjacent cysteine residues that resulted in the formation of 2 small loops (Angeli et al. 1999, Zhu et al. 2016, Pelosi et al. 2018, Zeng et al. 2019, Li et al. 2022).Research on the binding abilities showed that CSPs have good affinities with many chemicals, which later was identified that the 2 formed disulfide bridges do not place constraints on the scaffolding of the CSPs, so the proteins can enlarge their binding cavities and adapt to binding different components (Yi et al. 2014, Pelosi et al. 2018).The expression of CSPs was found to be mainly in the antennal; however, they also expressed in nonolfactory organs, which suggested CSPs in connection with different physical functions that are unrelated to chemoreception in insects (Pelosi et al. 2006, Gong, et al. 2007, Gu et al. 2012, Zhu et al. 2016, Martin-Blazquez et al. 2017, Fu et al. 2020).
The diamondback moth (DBM), Plutella xylostella L., is a destructive pest of cruciferous vegetables worldwide and causes enormous economic losses annually (Furlong et al. 2013).Plutella xylostella relies on the olfactory system to percept the volatile compounds released by host plants to select food sources and oviposition sites (Pivnick et al. 1994).Based on the antennal transcriptome analysis, 118 olfactory genes belonging to 6 chemoreception gene families were identified in P. xylostella L., including 54 ORs, 24 OBPs, 15 CSPs, 2 SNMPs, 7 gustatory receptors (GRs), and 16 IRs (Yang, et al. 2017).Furthermore, other 15 putative OBP genes were identified from its genome and transcriptome sequences of P. xylostella (Cai et al. 2021).Other 2 putative CSPs were identified from our previous unpublished data.However, only the functions of PxylCSP1 and PxylCSP11 have been reported among the 17 identified CSPs (Liu et al. 2010, Yi et al. 2014, Fu et al. 2020).Previous study showed that PxylCSP1 has a remarkable ability to bind the oviposition inhibitor, Rhodojaponin-III (R-III) (Liu et al. 2010).PxylCSP11, which mainly expressed in the antennae, has good affinities with special volatiles of cruciferous vegetables, phenethyl isothiocyanate and allyl isothiocyanate, the sex pheromone component, (Z)-11-hexadecenyl acetate, and other volatiles like α-terpineol, 2,4-dimethylheptane, α-terpinene, and phenethyl alcohol (Fu et al. 2020).Hence, we reasoned that PxylCSP11 also plays an essential role in feeding, mating, and oviposition sites selection.However, the detailed functional characteristics of other CSPs in P. xylostella still remain elusive so far.
Herein, we identified a new CSP of P. xylostella, PxylCSP18.The potential role in chemoreception and host plant recognition in P. xylostella L will be revealed and characterized.The findings of the study will be of great significance in enhancing our understanding of the olfactory system of P. xylostella.

Plutella xylostella Rearing
The larvae and pupae of P. xylostella were collected and reared in the Insect Neurobehavioral and Sensory Biology Laboratory, in Shanxi Agricultural University, Taigu Campus.The P. xylostella was maintained in a growth chamber at 25 ± 1 °C and 70 ± 5% relative humidity (RH) under a 14-h light (L): 10-h dark (D) photoperiod.P. xylostella larvae were fed with fresh cabbage (Brassica oleracea L.) and adults were supplied nutrition on absorbent cotton soaked with 10% honey solution.

Protein Sequence, Domain Identification, Phylogenetic Analysis, and Tertiary Structure Prediction
The amino acid sequences of the PxylCSP18 were retrieved from the NCBI database (https://www.ncbi.nlm.nih.gov/).Domains were predicted by searching InterPro (http://www.ebi.ac.uk/interpro/ search/sequence/) and the annotation of the amino acid sequence in the NCBI database.Homologs of PxylCSP18 were searched using the BlastP program in the NCBI database.The phylogram was built by the neighbor-joining method using MEGA 5.10 after aligning the selected CSP protein sequences using ClustalW.The tertiary structure of PxylCSP18 was developed by homology modeling (Swiss-Model: https://swissmodel.expasy.org/)based on the CSPsg4 of locust Schistocerca gregaria (Forskáhl) (Orthoptera: Acrididae).

RNA Extraction, cDNA Synthesis, and Quantitative Real-Time PCR
The total RNA of different tissue samples was isolated using TriPure Isolation Reagent (Roche, Switzerland) and purified by Direct-zol RNA Miniprep (Zymo Research, USA) according to the manufacturer's instructions.The extracted total RNA was quantified by spectrophotometric analysis, and the integrity was verified by agarose gel electrophoresis.cDNA was synthesized from the purified total RNA using Transcriptor First cDNA Synthesis Kit (Roche, Switzerland).
The quantitative real-time PCR (qRT-PCR) assay of PxylCSP18 was performed on a Bio-Rad CFX Connect Real-Time Detection System (Bio-Rad, California, USA) using the 2 × SG Fast qPCR Master Mix (Sangon, China).The gene of ribosomal protein S4 (Rps4) was used as an endogenous control (Fu et al. 2020).The results were evaluated using a relative quantitative method (2 −ΔΔCt ).All analyses were performed with 3 biological replicates.The primers used in qRT-PCR are listed in Table 1.The R 2 values of the standard curves were over 0.980 and the calculated amplification efficiency was 90%-110%.These indicated that the qRT-PCR were done in optimal conditions.All data were analyzed through a 1-way analysis of variance (ANOVA) followed by Tukey's honest significant difference (HSD) test (P < 0.05) in SPSS Statistics 17.0 software (SPSS Inc., Chicago, IL).

Double-Stranded RNA Synthesis and RNA Interference
The PxylCSP18 target fragment for RNA interference (RNAi) was amplified by PCR using the synthesized cDNA as template, and the primers were listed in Table 1.Both ends of the target fragment were added with T7 promoter sequences.After ensuring the bands of the PCR products were at the correct positions on the agarose gel, the specificity of the fragments was further confirmed via sequencing.The PCR products were purified using the Gel Extraction Kit (Promega, Madison, WI).The purified products were used to synthesize doublestranded RNA (dsRNA) with the T7 RiboMAX TM Express RNAi System (Promega, Madison, WI) according to the manufacturer.The purified dsRNA was quantified using spectrophotometry analysis, and the purity and integrity of dsRNA were verified using agarose gel electrophoresis.Finally, the purified dsRNA was then stored at −80 °C.The DNA fragment for the synthesis of dsGFP was amplified by PCR using our previously constructed reporter plasmid containing the open reading frame of GFP as a template (Ma et al. 2020), and the primers were listed in Table 1.dsGFP was also synthesized with the purified PCR products, as described above, to serve as an RNAi control.
The pupae the day before eclosion were collected and used for RNA interference.The pupae were anesthetized on ice and then injected with 1 μl of dsRNA (10 μg/μl) at the dorsal site of the abdomen using a Nanoject III micro-injector (Drummond Scientific, Broomall, PA) equipped with glass capillaries prepared using a P-97 micropipette puller (Sutter Instrument Co., Novato, CA).The antennae of day-2 unmated female and male adults were collected to detect the efficiency of RNAi-mediated knockdown of PxylCSP18 through qRT-RNA.Three independent biological replicates were performed.

Expression and Purification of Recombinant PxylCSP18
The coding region of mature PxylCSP18 was cloned from the synthesized cDNA with the specific primers, in which an EcoR V and an Xho I restriction sites were introduced (Table 1).The amplified product was cloned into the pET-32a vector (Novagen, USA), followed by transformation into the competent E. coli BL21 (DE3) (Tiangen, China).The expression of PxylCSP18 in the bacteria was induced by 0.6 mM isopropyl-beta-d-thiogalactopyranoside (IPTG) overnight.Subsequently, the bacterial cells were collected through centrifugation and resuspended into phosphate-buffered saline (PBS) solution, then homogenized through sonication on ice using an ultrasonic processor (Sonics Vibra-Cell, USA).PxylCSP18 was purified from the soluble faction using a Ni 2+ -NTA column (Roche, Switzerland) through a stepwise elution with 20, 50, 100, 150, and 500 mM imidazole in PBS.The fractions containing purified PxylCSP18 were collected and dialyzed against 20 mM Tris-HCl buffer containing 50 mM NaCl and 2 mM CaCl 2 (pH 7.4) overnight, followed by digestion by recombinant enterokinase (Solarbio, China) for 12 h to cleave the fragment containing Trx, His, and S tags, which led to PxylCSP18 lose the affinity for the Ni 2+ -NTA column.After repurified on Ni 2+ -NTA column, highly purified recombinant PxylCSP18 was acquired, which was then dialyzed against 20 mM Tris-HCl (pH 7.4).Finally, the PxylCSP18 was concentrated through ultrafiltration using a centrifugal device (3 kDa, Merck Millipore, USA), and the concentration was determined using the BCA Protein Assay Kit (Sangon, China).The purified recombinant PxylCSP18 was verified by peptide mass fingerprinting (Sangon Biotech, China).

Fluorescence Competitive Binding Assays
The fluorescence competitive binding assays were performed as described previously (Fu et al. 2020, Hua et al. 2021) with some modifications.Briefly, the emission spectra of the fluorescent probe, N-phenyl-1-naphthylamine (1-NPN, Sigma-Aldrich, USA), were recorded from 350 to 600 nm with 5 nm width of the emission slits and the excitation wavelength was 337 nm.To measure the affinity of 1-NPN to PxylCSP18, 1 µM protein in 20 mM Tris-HCl buffer (pH 7.4) was titrated with 1-NPN to final concentrations ranging from 1 to 17 µM in 1 cm light path quartz cuvette.After incubation for 3 min, the fluorescence intensity for each test was detected on an RF-5301 fluorescence spectrophotometer (Shimadzu, Japan).The average data of the recorded fluorescence intensity at the maximum emission wavelength were linearized using the Scatchard equation, and the dissociation constants for 1-NPN were then calculated (Sideris et al. 1992, Hua et al. 2021).Sixteen ligands, including 3 sex pheromone components ((Z)-11-hexadecenal, (Z)-11-hexadecenyl acetate, and (Z)-11-hexadecenyl alcolol) and some host volatiles (terpene, ketone, aldehyde, alcohol, and ester volatiles), were detected the affinities of PxylCSP18.The final concentrations for each ligand ranging from 1 to 17 µM were added into the mixed solution of 1-NPN probe (1 µM) and PxylCSP18 (1 µM).The acquired data were calculated from the Scatchard plots for dissociation constants (K d ) and the curves, assuming PxylCSP18 was active completely and bound to the ligands in a 1:1 ratio at saturation.K d of the competitor was calculated according to the following equation Underline showed the EcoR V and Xho I restriction enzyme sites.
a Only gene-specific parts of the primers are listed.These are preceded by the T7 adaptor TAATACGACTCACTATAGGG for dsRNA synthesis.concentration of ligand halving the initial value of fluorescence, [1-NPN] is the free concentration of 1-NPN and, K 1-NPN is the dissociation constant of the protein/1-NPN complex (Hua et al. 2021).

Y-Tube Olfactometer Bioassays
The responses of the day-2 unmated female and male adults to the volatile compounds were performed using a glass Y-tube olfactometer with a central glass tube (5 cm long × 2.5 cm ID) and 2 lateral glass arms (15 cm long × 2.5 cm ID) with a 75° inside angle as described previously (Yan et al. 2023), with some modifications.Before bioassays, all glasswares were cleaned and washed with absolute alcohol and dried at 100 °C for 5 min.A moistened and activated charcoal-filtered airflow (350 mL/min) was pumped into each source container using an atmospheric sampling instrument (QC-1S, Beijing Institute of Labor Protection Science, Beijing, China).The behavioral response of male and female adults of P. xylostella to trans-2-hexen-1-ol, isopropyl isothiocyanate, myrcene, linalool were then observed.Each component was diluted with paraffin oil to 10 μg/μl.For each chemical, an aliquot of 10 μl was applied to a strip of filter paper (0.5 × 5 cm) and placed in 1 source container.A filter paper strip loaded with the same volume of paraffin oil and placed in the second source container was used as a control.Each test male or female moth was released at the entrance of the central glass tube of the Y-tube olfactometer and observed for 5 min.A moth was considered to have made a "choice" when it moved more than 10 cm into either arm and remained there for at least 30 s; "no choice" was recorded when the moth did not make a choice within 5 min.The positions of the arms were reversed after 5 insects to avoid positional bias.The experimental device was cleaned after every 10 insects by washing it with absolute acetone and followed by heating at 100 °C for 5 min.Each concentration of compound stimulus was tested with 30 insects, which successfully made a "choice."The choice index was calculated as the number of responses of moths to control arms divided by the total number of test moths.

Expression Pattern of PxylCSP18
The expression levels of PxylCSP18 in different tissues (antennae, heads without antennae, thoraxes, abdomen, legs, and wings) of female and male adult moths were analyzed by qRT-PCR.The results showed that PxylCSP18 was expressed in all the tested tissues of females and males, and the highest expression level was found in antennae (Fig. 2, F (11, 24) = 1087, P < 0.001).The expression level of PxylCSP18 in male antennae was obviously higher than that in female antennae (Fig. 2).Additionally, the expression of PxylCSP18 in heads (without antennae) was higher than that in other tissues, but it was much lower than that in antennae, which suggested that PxylCSP18 mainly plays a role in antennae (Fig. 2).

The Purification of the Recombinant PxylCSP18
To further investigate the physiological function of PxylCSP18, the recombinant PxylCSP18 was expressed and purified from E. coli (DE3).The recombinant PxylCSP18 was expressed as a fusion protein with a hexahistidine and a Trx tags in the N-terminus to facilitate its correct folding and purification.The induction and purification of recombinant PxylCSP18 with Trx-hexahistidine tags was illustrated as Fig. 3A.After digestion with enterokinase, the PxylCSP18 lost the Trx-hexahistidine tags.After purified on Ni 2+ -NTA column, highly purified recombinant PxylCSP18 without tags was acquired (Fig. 3B).The purified recombinant protein was verified by peptide mass fingerprinting as hypothetical OS-D domain protein, which meant the identified PxylCSP18 (XP_011563205.3) (Fig. 3C and Supplementary form 1).
The molecular docking of PxylCSP18 and γ-terpinene based on homology modeling was performed to further investigate the combination of PxylCSP18 and ligands.The PxylCSP18 had a strong binding affinity to γ-terpinene and the binding free energy of proteinligand complex was -5.7 kcal/mol.The binding pocket was formed by α1, α2, and α3 and located near the N-term of PxylCSP18 (Fig. 5A and B).Concretely, Pi-Pi Stacked was formed by γ-terpinene-Phe: 31; Pi-Alkyl or Alkyl bonds were formed by γ-terpinene-Tyr: 28, γ-terpinene-Ile: 42, and γ-terpinene-Pro: 58; van der Waals forces were formed by γ-terpinene-Tyr: 46 and γ-terpinene-Asp: 59 (Fig. 5B and C).These forces were the main forces for PxylCSP18 and γ-terpinene.Hence, we here demonstrated that PxylCSP18 had a strong binding ability to many repellent and attractive plant volatiles on different levels (Table 2, Figs.4B and 5).These data suggested PxylCSP18 plays an important role in the unsuitable host avoidance and food sources/host detection.

Behavioral Responses of P. xylostella Adults to Volatile Compounds After Knockdown the Expression of PxylCSP18
After injected dsCSP18 for 3 days, the expression of PxylCSP18 decreased by approximately 70% and 48% in the unmated male (Fig. 6A) and female (Fig. 6B) adults (day 2), respectively.Trans-2hexen-1-ol and isopropyl isothiocyanate were randomly selected as the attractive volatile compounds and myrcene and linalool were randomly chosen as the repellent volatile compounds, to explore the behavioral responses of P. xylostella adults to volatile compounds after knocked down the expression of PxylCSP18.The attractive effects of trans-2-hexen-1-ol and isopropyl isothiocyanate to P. xylostella male adults were significantly decreased after knocked down the expression of PxylCSP18 (Fig. 6C).The attractive effects of isopropyl isothiocyanate and the repellent effects of linalool to P. xylostella female adults were significantly weakened after knocked down the expression of PxylCSP18 (Fig. 6D), while the repellent effects of myrcene and linalool to P. xylostella male adults, and attractive effects of trans-2-hexen-1-ol and repellent effects of myrcene P. xylostella female adults decreased inconspicuously after knocked down the expression of PxylCSP18 (Fig. 6C and D).

Discussion
CSPs are abundant in the sensillum lymph, and they are mainly responsible for capturing and transporting odor molecules from the external environment to ORs (Pelosi et al. 2006).Thus, molecular function studies on CSPs are of great significance to reveal chemical communication mechanism between insects and host plants.
In this study, the PxylCSP18 was cloned from P. xylostella, and the properties of the PxylCSP18 were discussed.The PxylCSP18 has a signal peptide in the N-terminal and 4 highly conserved cysteines  in the OS-D domain, which are the typical common characteristics of insect CSPs (Zhou et al. 2006, Zhang and Lei 2015, Ali et al. 2019).The tertiary structure and phylogenetic analyses further confirmed that the PxylCSP18 was a typical CSP.PxylCSP18 was clustered in a group with other lepidopterous CSPs, indicating the conservation of phylogeny in insect CSPs.According to antennal transcriptome analysis, GmolCSP11 is highly expressed in female antennae of G. molesta (Li et al. 2015).PxylCSP18 is orthologous to GmolCSP11 with 70% identity in amino acid sequence.Therefore, combined with the result of this study, we speculate that PxylCSP18 may have functionality similar to GmolCSP11.
The expression profiles of CSPs in different tissues suggest functional differentiation of CSPs (Yang et al. 2014).The analysis of the relative expression in various tissues of P. xylostella showed that PxylCSP18 was mainly expressed in antennae and the expression level in male antennae was obviously higher than that in female antennae.Generally, CSPs were mainly isolated from sensory organs of insect (Pelosi et al. 2006).Antennae and maxillary palps are the main olfactory organs of insect, and they are covered with kinds of porous sensilla (Wicher and Miazzi 2021).PxylCSP18 was also found to have a high expression in the head (without antennae) of P. xylostella, but it was much lower than that in antennae.Therefore,  PxylCSP18 might play a role in other olfactory organs of P. xylostella, such as the maxillary palps, except for antennae.

Schroder
Insect CSPs are usually thought to contribute to the transport of ligand from the sensilla cuticle to receptors in olfactory neurons (Yi et al. 2014).Hence, based on the results discussed above, PxylCSP18 might be involved in the perception of host volatiles and sex pheromones (Zhang et al. 2014, Zeng et al. 2018).Furthermore, the binding characteristics analysis showed that PxylCSP18 had strong binding affinities with the 14 plant volatiles.Meanwhile, the repellent and attractive effects of the adults to the repellent and attractive volatile compounds were decreased after knockdown of PxylCSP18.These results together revealed that PxylCSP18 play an important role in olfactory perception by binding and transporting plant volatiles (Peng et al. 2017, Hua et al. 2021, Li et al. 2021, Wang et al. 2021).
In conclusion, by discussing the characteristics and functions of PxylCSP18 in P. xylostella, this study provides new information for investigating the physiological role of insect CSPs.It will contribute to understanding the underlying the olfactory communication mechanism of P. xylostella.

Fig. 2 .
Fig. 2. The tissue expression levels of PxylCSP18 in the female and male P. xylostella adults.The tissues included antennae (An), heads without antennae (He), thoraxes (Th), abdomens (Ab), legs (Le), and wings of the female and male adults.The values shown were the mean (±SEM) of 3 independent experiments.Lowercase letters (a, b,c, d) above the error bars denoted significant differences in the tissues at P < 0.05.P values were determined by ANOVA followed by Tukey's honestly significant difference test.

Fig. 3 .
Fig. 3. Expression and purification of recombinant PxylCSP18 analyzed by SDS-PAGE.A) M: protein molecular mass markers.Lane 1: total proteins from uninduced E. coli cells; lane 2: total proteins from induced E. coli cells; lane 3: soluble proteins from induced E. coli cells; lane 4: purified fusion protein before enterokinase digestion (pointed by the black arrow).B) Lane 1: purified PxylCSP18 (pointed by the black arrow).C) The mass spectrometry results of the protein band excised from the gel in (B).

Fig. 4 .
Fig. 4. The binding affinities of PxylCSP18 with various ligands.A) Binding curve of the PxylCSP18 with fluorescent probe1-NPN and the relative Scatchard plot analysis.B) Competitive binding curves of PxylCSP18 with various odorant ligands.
. is the abbreviation for not detected, which means that no significant binding was detected in the assay.a Means the plant volatiles, which are attractive to P. xylostella, are detected in cruciferous vegetables(Reddy and Guerrero 2000a, Han et al. 2001, Dai et al. 2008, Li et al. 2012, Yan et al. 2023).b Means the plant volatiles, which are repellent to P. xylostella, are detected in nonhost plant(Song et al. 2022).

Table 2 .
Binding affinities of PxylCSP18 with volatile ligands